Lysosomal storage disorders are a group of inherited disorders characterized by deficiencies in specific enzymes in the body, which results in the body's inability to break down metabolic substances. As an example, Fabry disease is a lysosomal storage disorder seen in one out of every 40,000 people. It is caused by a deficiency in the enzyme alpha-galactosidase which results in the body's inability to break down specific fatty substances called globotriaosylceramides. A second example is Gaucher disease, a lysosomal storage disorder caused by an inability to break down fatty substances or lipids called glucosylceramides (also called glucocerebrosides). Individuals with Gaucher disease do not make glucocerebrosidase, an enzyme needed to break down these fatty substances. These fatty substances then accumulate in cells of the liver, spleen, and bone marrow. A third example is Pompe disease, a lysosomal storage disorder caused by a deficiency in the enzyme acid alpha-glucosidase, which is needed to break down certain sugars called glycogen. When the enzyme acid alpha-glucosidase is missing, glycogen accumulates in various tissues and organs in the body.
Lysosomal storage disorders are, for the most part, childhood disorders although some manifest in adulthood. In most of them, patients are normal at birth and have progressive neurological deterioration beginning at some later time. The clinical phenotype depends on the type and severity of the biochemical defect. Some of these lysosomal disorders, such as Pompe disease and Krabbe disease, manifest primarily in infancy. There have been ongoing efforts in developing methods to detect such disorders before the onset of clinical symptoms so that therapeutic interventions can be initiated.
Most of the screening tests for a lysosomal storage disorder (LSD) offered today are based on the mass spectrometry method and materials described by Zhang et al. (Methods Mol Biol. 2010; 603: 339-350). This method, for six LSDs (Pompe, Krabbe, Niemann-Pick A&B, Fabry, Gaucher, and MPS-1), conducts the six enzyme activity assays in six separate vessels using six separate conditions. Multiplex assays for the same disorders have been described (Scott et al, J Pediatr. 2013 August; 163(2):498-503; Spazil et al, Clin Chem. 2013 March; 59(3):502-11). Multiplex methods require less laboratory labor and reagents but it is generally more difficult to produce assay results of similar quality to non-multiplex methods where the assay conditions need to represent a compromise that supports adequate activity of the target enzymes while suppressing any confounding processes. Various enzyme activity enhancers and suppressors are added to multiplex buffers to support an optimized condition that allows each of the targeted enzymes to act measurably on its specific artificial substrate.
Oleic acid has been identified as an enhancer for the galactocerebrosidase (GALC) enzyme, for which low activity is associated with Krabbe disease (Zhang et al, ibid; Zhang et al Clin Chem. 2008; 54(10): 1725-1728; Li et al, Clin Chem. 2004; 50(10): 1785-1796). This acid has been added to buffers for Krabbe disease screening. However, oleic acid is very difficult to dissolve in aqueous buffers and the resulting varying concentration in assay buffer has led to assay results with poor reproducibility. For this reason, most published protocols for Krabbe disease screening omit oleic acid as the reproducibility problems outweigh any potential performance advantages from enhanced enzyme activities.
Thus, there is a continuing need for improving the methods and compositions for detecting lysosomal disorders.